In many, if not most, plant species, the development of hybrid cultivars is highly desired because of their generally increased productivity due to heterosis: the superior performance of hybrid individuals compared with their parents (see, e.g., Fehr (1987) "Principles of Cultivar Development, Volume 1: Theory and Technique", MacMillan Publishing Company, New York; Allard (1960) "Principles of Plant Breeding", John Wiley and Sons, Inc., New York).
The development of hybrid cultivars of various plant species depends upon the capability to achieve almost complete cross-pollination between parents. This is most simply achieved by rendering one of the parent lines male-sterile (i.e., with pollen being absent or nonfunctional); for example, by manually removing the one parent's anthers or by providing the one parent with naturally occurring cytoplasmic or nuclear genes that prevent anther and/or pollen development and/or function, using classical breeding techniques (for a review of the genetics of male-sterility in plants, see Kaul (1988) "Male Sterility in Higher Plants", Springer Verlag, New York).
For hybrid plants where the seed is the harvested product (e.g., corn and oilseed rape), it is, in most cases, also necessary to ensure that fertility of the hybrid plants is fully restored. In plants in which the male-sterility is under genetic control, this requires the use of genes that can restore male-fertility. Hence, the development of hybrid cultivars is mainly dependent on the availability of suitable and effective sterility and restorer genes.
Endogenous nuclear loci are known for most plant species that contain genotypes which effect male-sterility, and generally, such loci need to be homozygous for particular recessive alleles in order to result in a male-sterile phenotype. The presence of a dominant male-fertile allele at such loci results in male-fertility.
Recently, it has been shown that male-sterility can be induced in a plant by providing the plant with a nuclear male-sterility genotype that includes a chimaeric male-sterility gene comprising a DNA sequence (or male-sterility DNA) coding, for example, for a cytotoxic product (such as an RNase) and under the control of a promoter which is predominantly active in selected tissue of the plant's male reproductive organs. In this regard, tapetum-specific promoters, such as the promoter of the TA29 gene of Nicotiana tabacum, have been shown to be particularly useful for this purpose (Mariani et al (1990) Nature 347:737; European patent publication ("EP") 0,344,029). By providing the nuclear genome of the plant with such a male-sterility gene, an artificial nuclear male-sterility locus is created containing the artificial male-sterility genotype that results in a male-sterile plant.
In addition, it has been recently shown that male-fertility can be restored to such a nuclear male-sterile plant with a chimaeric fertility-restorer gene comprising another DNA sequence (or fertility-restorer DNA) that codes, for example, for a protein that inhibits the activity of the cytotoxic product or otherwise prevents the cytotoxic product from being active at least in the selected tissue of the plant's male reproductive organs (EP 0,412,911). For example, the barnase gene of Bacillus amyloliquefaciens codes for an RNase (Barnase) which can be inhibited by a protein (Barstar) that is encoded by the barstar gene of B. amyloliquefaciens. Hence, the barnase gene can be used for the construction of a chimaeric male-sterility gene while the barstar gene can be used for the construction of a chimaeric fertility-restorer gene. Experiments in different plant species (e.g., oilseed rape) have shown that such a chimaeric barstar gene can fully restore the male-fertility of male-sterile lines in which the male-sterility was due to the presence of a chimaeric barnase gene (EP 0,412,911: Mariani et al (1991) Proceedings of the CCIRC Rapeseed Congress, Jul. 9-11, 1991 Saskatoon, Saskatchewan, Canada; Mariani et al (1992) Nature 357:384). By coupling a marker gene, such as a dominant herbicide resistance gene (for example, the bar gene coding for phosphinothricin acetyl transferase (PAT) that converts herbicidal phosphinothricin to a non-toxic compound [De Block et al (1987) EMBO J. 6:2513]), to the chimaeric male-sterility and/or fertility restorer gene, breeding systems can be implemented to select for uniform populations of male-sterile plants (EP 0,344,029; EP 0,412,911).
The production of hybrid seed of any particular cultivar of a plant species requires the: 1) maintenance of small quantities of pure seed of each inbred parent; and 2) the preparation of larger quantities of seed of each inbred parent. Such larger quantities of seed would normally be obtained by several (usually two) seed-multiplication rounds, starting from a small quantity of pure seed ("basic seed") and leading, in each multiplication round, to a larger quantity of seed of the inbred parent and finally to a stock of seed of the inbred parent ("parent seed" or "foundation seed") which is of sufficient quantity to be planted to produce the desired quantities of hybrid seed. Of course, in each seed-multiplication round, larger planting areas (fields) are required.
In order to maintain and enlarge a small stock of seeds of male-sterile plants, it has been necessary to cross the parent male-sterile plants with normal pollen-producing parent plants. The offspring of such a cross will, in all cases, be a mixture of male-sterile and male-fertile plants, and the latter have to be removed from the former. With male-sterile plants containing an artificial male-sterility locus as described above, such removal can be facilitated by genetically linking the chimaeric male-sterility gene to a suitable marker gene, such as the bar gene, which allows the easy identification and removal of the male-fertile plants. EP 0,198,288 and U.S. Pat. No. 4,717,219, by comparison, describe methods for linking such marker genes (which can be visible markers or dominant conditional markers) to endogenous nuclear loci containing male-sterility genotypes.
However, even when suitable marker genes are linked to male-sterility genotypes, the maintenance of parent male-sterile plants still requires the removal from the field of a substantial number of plants. For instance, in systems using a herbicide resistance gene (e.g., the bar gene) linked to a chimaeric male-sterility gene, only half of the parent stock will result in male-sterile plants, thus requiring the removal of the male-fertile plants by herbicide spraying prior to flowering. In any given field, the removal of male-fertile plants effectively reduces the potential yield of hybrid seed or the potential yield of male-sterile plants during each round of seed multiplication for producing of parent seed. This is economically unattractive for many important crop species such as corn and oilseed rape. In order to minimize the number of male-fertile plants which have to be removed, male-fertile maintainer plants have been sought which, when crossed with a male-sterile parent plant, produce a minimum, preferably no, male-fertile offspring, thereby minimizing or avoiding altogether the need to remove such male-fertile offspring. To solve an analogous problem, U.S. Pat. Nos. 3,710,511 and 3,861,079 have described procedures for producing and maintaining a homogenous population of male-sterile plants by using specific chromosomal abnormalities that are differentially transmitted to the egg and the sperm in the plants.